EasySep Hu CB CD34 Pos Sel Kit II

STEMCELL Technologies' EasySep™ kit enables immunomagnetic positive selection of human CD34+ cells from fresh whole umbilical cord blood using a simple two-step procedure. The protocol first combines standard density gradient centrifugation with pre-enrichment of CD34+ cells using a RosetteSep™ Human Cord Blood CD34 Pre-Enrichment Cocktail, which cross-links unwanted mature cells to red blood cells to form dense immunorosettes. After centrifugation, the CD34+-enriched fraction is collected from the interface between the plasma and the density gradient medium.

In the second step, pre-enriched CD34+ cells are labeled with the EasySep™ isolation cocktail and magnetic EasySep™ Dextran RapidSpheres™. The labeled cell suspension is transferred to an EasySep™ magnet (sold separately) for positive selection; after a brief incubation, unwanted CD34− cells are poured off while the desired CD34+ cells remain in the tube. Density gradient centrifugation during pre-enrichment can also be performed in a SepMate™ tube, which contains an insert that prevents the density gradient medium and blood sample from mixing and eliminates the need to carefully layer the sample, reducing total separation time.

In a typical experiment, CD45+CD34+ HSPCs are enriched > 15-fold (from 0.6% to 10%) after RosetteSep™ pre-enrichment and > 200-fold (from 0.6% to 98%) after EasySep™ CD34+ selection. Based on cell separations with 15 different cord blood samples with a starting CD34+ cell purity of 0.4%, the average CD34+ cell purity is 6% after RosetteSep™ pre-enrichment and 91% after EasySep™ cell isolation, with the CD34+ cell content of the isolated fraction typically 91 ± 9% (mean ± SD) using the purple EasySep™ Magnet. Each kit contains reagents sufficient for processing up to 1000 mL of cord blood.